Reactive oxygen species (ROS) are involved in the hepatotoxicity of high concentrations of ethanol. A number of mechanisms such as decreased concentrations of mitochondrial GSH, inhibition of respiration and enhanced susceptibility to hypoxia have been invoked. The focus of this proposal is to examine the effects of ethanol on hepatic mitochondrial function mediated through nitric oxide (NO), reactive nitrogen species (RNS) and ROS. Previous studies and preliminary data have shown that a biphasic effect of NO can be demonstrated in mitochondria in terms of control of both respiratory function and the processes leading to cytochrome c release. Although many of the mitochondrial defects elicited by ethanol can also be caused by RNS a mechanistic link has not been examined. The concept developed in this proposal is that NO is converted to a mitochondrial toxin on ethanol consumption by reaction with ROS. Key findings in support of this hypothesis are 1) iNOS is induced on ethanol consumption in vivo and its product, NO, is a potent regulator of mitochondrial respiration 2) this is associated with increased tyrosine nitration-a marker of RNS 3) NO, in contrast, inhibits cytochrome c release from mitochondria, whereas RNS such as peroxynitrite, promotes release of this pro-apoptotic factor 4) chronic ethanol consumption leads to greater sensitivity of the hepatocyte to hypoxic stress. These data have led to the hypothesis that a critical mechanism contributing to alcohol hepatotoxicity is through NO-dependent modification of mitochondrial function. This concept will be tested by pursuit of the following Specific Aims in wild type and INOS knock out mice consuming ethanol: 1. Determine the effects of increased NO by chronic alcohol consumption on mitochondrial respiration, antioxidant capacity and ROS/RNS formation. 2. Determine the effect of chronic alcohol consumption on cytochrome c release and mitochondrial protein modification in isolated organelle and hepatocytes. 3. Determine the NO-dependent effects on mitochondrial protein synthesis and the response of hepatocytes to hypoxic stress after chronic consumption of ethanol.